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Applied and Environmental Microbiology, January 2003, p. 139-145, Vol. 69, No. 1
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.1.139-145.2002
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Thermostabilization of Bacterial Fructosyl-Amino Acid Oxidase by Directed Evolution

Ryoichi Sakaue* and Naoki Kajiyama

Research and Development Division, Kikkoman Corporation, Noda City, Chiba Prefecture 278-0037, Japan

Received 25 June 2002/ Accepted 21 October 2002

We succeeded in isolating several thermostable mutant fructosyl-amino acid oxidase (FAOX; EC 1.5.3) without reduction of productivity by directed evolution that combined an in vivo mutagenesis and membrane assay screening system. Five amino acid substitutions (T60A, A188G, M244L, N257S, and L261M) occurred in the most thermostable mutant obtained by a fourth round of directed evolution. This altered enzyme, FAOX-TE, was stable at 45°C, whereas the wild-type enzyme was not stable above 37°C. The Km values of FAOX-TE for D-fructosyl-L-valine and D-fructosyl-glycine were 1.50 and 0.58 mM, respectively, in contrast with corresponding values of 1.61 and 0.74 mM for the wild-type enzyme. This altered FAOX-TE will be useful in the diagnosis of diabetes.

* Corresponding author. Mailing address: Research and Development Division, Kikkoman Corporation, 399 Noda, Noda City, Chiba Prefecture 278-0037, Japan. Phone: 81-4-7123-5569. Fax: 81-4-7123-5968. E-mail: rsakaue{at}mail.kikkoman.co.jp.

Applied and Environmental Microbiology, January 2003, p. 139-145, Vol. 69, No. 1
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.1.139-145.2002
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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